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Ghrelin Prevents Levodopa-Inhibited Gastric Emptying in Rats
GE. Plasma acyl ghrelin levels were not altered at 30 min, 1 h and 2 h after iv injection of levodopa compared to basal levels and vehicle treated fasted rats. Conclusion: These data suggest that ghrelin can be used as a prokinetic agent to improve the levodopa-induced delayed GE and has the potential to improve delayed levodopa absorption in the small intestine which can result in response fluctuations in PD patients. Supported by The Michael J Fox Foundation for Parkinson's Research Su1722 Reduced Ghrelin Production Induced Anorexia After Gastric Ischemia and Reperfusion in the Rat Sachiko Mogami, Hidekazu Suzuki, Hitoshi Tsugawa, Juntaro Matsuzaki, Yoshio Kase, Toshifumi Hibi Background. The gastrointestinal tract is one of the most susceptible organs to ischemia. We recently reported post-ischemic alterations of the gastric motility after gastric ischemia and reperfusion (I/R), along with disruption of the interstitial cells of Cajal network and decrease of neuronal nitric oxide synthase-positive neurons (Neurogastroenterol. Motil. 2010, 22(5): 585-93). Ghrelin is a gastrointestinal peptide that stimulates growth hormone secretion and gastrointestinal motility. Ghrelin has been demonstrated to attenuate the mucosal injuries induced by gastric I/R (J. Gastroenterol. Hepatol. 22: 1791, 2007); however, the alterations of the eating behavior and ghrelin dynamics after gastric I/R have not yet been elucidated. The aim of the present study was to investigate the relationship between the ghrelin dynamics and food intake after gastric I/R. Materials and Methods. Seven week-old male Wistar rats were used after food deprivation. Under anesthesia, the celiac artery was clamped for 80 min, followed by 12 or 48 h reperfusion. The plasma ghrelin level was measured by ELISA. Real-time PCR of preproghrelin mRNA and immunohistochemical staining using anti-ghrelin antibody were performed after gastric I/R. Food intake was measured after gastric I/R. Ghrelin was administered intraperitoneally to elucidate whether exogenously administered ghrelin restore the reduced food intake after gastric I/R. Results. The plasma acyl-ghrelin levels at 12 h and 48 h after I/R were significantly lower than those in the sham-operated rats at the corresponding time-points (sham 12 h; 64.5 ± 13.5 fmol/ mL, I/R 12 h; 46.3 ± 9.25 fmol/ mL, sham 48 h; 97.2 ± 30.3 fmol/ mL, I/R 48 h; 70.9 ± 18.4 fmol/ mL). The plasma desacylghrelin levels at 12 h and 48 h after I/R were also lower than those in the sham-operated rats at the corresponding time-points (sham 12 h; 834 ± 137 fmol/ mL, I/R 12 h; 663 ± 113 fmol/ mL, sham 48 h; 1092 ± 150 fmol/ mL, I/R 48 h; 835 ± 187 fmol/ mL). The expression levels of preproghrelin mRNA were significantly reduced at 12 h and 48 h after I/R as compared with the levels in the sham-operated rats at the corresponding time-points. The count of ghrelin-immunoreactive cells was also decreased at 12 h after I/R, but recovered by 48 h. However, the mucosal injuries persisted throughout the observation period. Cumulative food intake was reduced at 12hr and 48 h after gastric I/R as compared with that in the sham-operated rats. Intraperitoneal administration of ghrelin restored the decreased food intake of the rats observed after I/R. Conclusion. The consistent decrease of the plasma ghrelin levels is considered to be attributable to the gastric mucosal injuries. The decreased plasma ghrelin levels may have induced the decrease in the food intake after gastric I/R, as it was restored by exogenous ghrelin.
Su1720 A Novel eADO Biosensor Microelectrode Array (MEA) for Real-Time Electrochemical Detection of eADO Release From Mouse or Human Gut Iveta Grants, Aurosree Bhowmik, Niyati Dhand, Bradley Needleman, Alan Harzman, Jacqueline E. Wunderlich, Fievos L. Christofi Background and Aims: Endogenous adenosine (eADO) release inhibits neurotransmission, modulates gut mucosal reflexes and protects against colitis. ADO is “a danger signal” in diseases and its release is estimated to reach 10-100 fold higher levels than normal. Little is known about release mechanism(s) of eADO and accurate measurement of ADO in extracellular fluid in response to different stimuli is of critical importance in health and disease. The goal was to develop and test a new eADO biosensor to monitor real-time changes of eADO release in gut mouse or human tissues. Methods: An enzyme linked eADO biosensor / microelectrode array (MEA) FAST16 mk electrochemical recording system was used to monitor changes. The MEA tip sensor has S1 and S2 sites to measure local release of eADO and eINO respectively - the eADO platinum(pt) site S1 is coated with 3 enzymes starting with adenosine deaminase (ADA+NP+XO) to sequentially break down ADO to H202 for detection (S2/eINO site lacks ADA). H202 is oxidized on the pt-surface and resulting current is quantified. Standard curves to ADO or INO (0.5-100μM) are used to calculate release (from current of 0.05nA-1.5nA); an ADA inhibitor EHNA (5μM) competitively inhibited ADO responses. Submucosal plexus with mucosa (M- ENSSMP) or ENSSMP is dissected from human sigmoid colon or jejunum (polyp, Roux-en-Y surgeries) or mouse colon. Recordings are made in a perfusion chamber in Krebs buffer at 37°C; 3-5 tissues/ condition are used. A Narishigi (or Eppendorff) micromanipulator visually guided by a stereomicroscope (zoom 1-40X) is used to position the MEA tip on or near the mucosal surface or ganglion (G) to measure rapid release of eADO (or eINO at 40Hz) in a 200μmspace. Results: In mouse or human ENSSMP high K+ depolarization or high Mg2+/low Ca2+ solutions stimulate release of eADO by 0.5-5μM. DMPP (1-10μM) evokes a TTX-insensitive elevation in eADO of 0.25-0.5μM in ENSSMP. Ischemia, 100μM histamine or blockade of nucleoside transport by 2μM NBTI elevates eADO by 0.25-1.0μM. DSS colitis (3%, 7 days) causes low level oscillations in basal eADO release. In human ENSSMP LSM Fluo-4/Ca2+ imaging revealed that NBTI blocks slow synaptic transmission (sEPSCaT) evoked by fiber tract stimulation (n=12 neurons/G). ADO inhibits and ADA (2u/ml) augments - sEPSCaTs (n=20, p<0.01). Touch or mucosal compression (MS) increased eADO release 10-20μM in humucosa >> huENSSMP). DSS colitis augmented eADO release to MS or K+ depolarization in mucosa and caused prolonged, high amplitude/cyclical responses lasting > 20 min (control=5 min). Conclusions: The enzyme-linked eADO biosensor is a suitable technique to monitor realtime release and quantification of eADO in normal or inflamed gut. Release is TTX-insensitive. Further study is needed to distinguish between H202 from eADO release and H202 from inflammation (DK44179-15;DK44179-14S/ARRA).
Su1723 Impaired Gastric Emptying in Gastroesophageal Reflux Disease Rat Models is Caused by a Reduced Response to Ghrelin Hiroshi Takeda, Shuichi Muto, Nobuhiko Oridate, Shunsuke Ohnishi, Koji Nakagawa, Chiharu Sadakane, Yayoi Saegusa, Miwa Nahata, Tomohisa Hattori, Masahiro Asaka Background/Aim: Adequate conclusions on the association between the onset and progression of gastroesophageal reflux disease (GERD) and gastrointestinal motility have not yet been reached; however, some enterokinesis-promoting agents that may reverse the mucosal damage of GERD have been recognized. Furthermore, there is speculation that reduced gastric emptying in GERD patients is partially involved in acid reflux. Ghrelin is a strong appetite-stimulating hormone produced in the stomach, and is known to have a powerful enhancing effect on gastrointestinal motility. We have clarified the involvement of ghrelin in gastric emptying in a GERD rat model. Method: The GERD model was induced in Wistar rats (Omura N et al. Scand J Gastroenterol 1999; 34:948-953). Rat acyl ghrelin was administered intravenously to the GERD model rats, and the effect on gastric emptying using a semi-solid meal method and feeding were examined by comparison with a sham group. Gastric motor activity in the antrum was evaluated using a strain gauge force transducer. Furthermore, in order to study the effects on ghrelin secretory function and signal transmission in the GERD model rats, the ghrelin levels in the blood and expression of appetite-related genes in the hypothalamus were also examined. Results: In comparison with the sham group, gastric emptying was significantly reduced in the GERD model rats (sham rats, 93.4 ± 2.2 %; GERD rats, 59.2 ± 10.9 %; p < 0.05 vs sham rats). In addition, the gastric antral motility index and frequency were reduced in the GERD model rats (Figure).Intravenous (IV) administration of rat acyl ghrelin significantly enhanced gastric emptying in the sham group, but not in the GERD model rats(Table). Furthermore, while IV administration of rat acyl ghrelin enhanced gastric antral motility in the sham group rats, it showed no effect on gastric antral motility in the GERD model rats. Similarly, IV administration of ghrelin to the sham group rats significantly increased feeding (saline, 1.0 ± 0.2 g/h; ghrelin, 1.8 ± 0.2 g/h; p < 0.05), but no effect was observed in the GERD model rats (saline, 0.6 ± 0.1 g/h; ghrelin, 0.4 ± 0.2 g/h). In addition, there was no difference in the plasma GH levels between the two groups of rats. In the GERD model rats, plasma ghrelin levels, hypothalamic NPY/AGRP mRNA expressions were also high compared to the levels in the sham group rats. Conclusion: In the GERD model rats, the ghrelin signal transmission, which may mediate gastric emptying, is reduced. Peripheral ghrelin signals are transmitted to the NPY/AGRP neurons, suggesting the possibility that signals are interrupted after transmission. Table The effect of intravenous administration of rat acyl ghrelin on gastric emptying.
Su1721 Ghrelin Prevents Levodopa-Inhibited Gastric Emptying in Rats Lixin Wang, Andreas Stengel, Miriam Goebel, Marie-Francoise Chesselat, Yvette Tache Background: Levodopa, a precursor of dopamine, is a traditional drug taken orally to treat symptoms of Parkinson's disease (PD). However, it induces delayed gastric emptying (GE), which dampens its absorption in the small intestine associated with motor fluctuation. Levodopa has been administered by different routes in rats and more variations in plasma levels occurred after oral administration compared to intravenous (iv) or intraperitoneal (ip) injection. Ghrelin is a potent prokinetic hormone involved in the regulation of gastric phase III-like contractions and its agonists are in clinical trials for treatment of post-operative ileus. Dopamine and ghrelin have opposite effects on gastrointestinal motility. Aim: To investigate whether ghrelin blocks levodopa inhibitory effect on GE. Methods: GE was determined by the phenol red method in fasted rats 20 min after (1) levodopa (5 or 15 mg/kg) or vehicle (0.3 ml of 0.2N HCl and 7% NaHCO3 at 4:1.5 with 1% ascorbic acid, pH 7) co-administered intragastrically (ig) with a non-nutrient viscous phenol red solution (1.2 ml/rat); (2) ip injection of levodopa (15 mg/kg) or vehicle (0.5 ml/rat) 10 min before ig viscous phenol red solution; (3) iv injection of ghrelin (30 or 100 μg/kg) or vehicle (saline 0.2 ml/rat) 10 min before ig levodopa and viscous phenol red solution; (4) subcutaneous (sc) injection naltrexone (μ opioid receptor antagonist as a control agent, 1 mg/kg) or saline (0.5 ml/rat) 30 min before ig levodopa and viscous phenol red solution. Plasma acyl ghrelin levels after iv levodopa (5 mg/kg) were assessed by radioimmunoassay. Results: Levodopa administered ig dose-dependently decreased GE in fasted rats (5 or 15 mg/kg: 41.0 ± 5.0 or 23.0 ± 4.8% respectively vs. 59.9 ± 3.5% in vehicle, P < 0.05), and the inhibition was more robust after ip injection (15 mg/kg: 8.0 ± 2.5%, P < 0.05). Pretreatment with iv ghrelin completely blocked the ig levodopa-inhibited GE (53.2 ± 3.0 or 59.2 ± 2.3% at 30 or 100 μg/kg vs. 30.1 ± 4.4% with iv saline, P < 0.05) compared to iv saline plus ig vehicle (60.0 ± 5.2%, P > 0.05). Ghrelin increased basal GE (72.9 ± 2.3%) but not significantly compared with saline (P > 0.05). Naltrexone injected sc did not influence the ig levodopa-induced delayed
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AGA Abstracts
AGA Abstracts
the nuclear proteins binding to the TPH1 promoter. ZBP-89Δcolon mice were infected with Salmonella typhimurium (S. typhi) at 107 cfu for 3 days. RESULTS: Although ubiquitously expressed, immunofluorescence confirmed high expression of ZBP-89 protein with serotonin in both mouse and human gut EC cells. ZBP-89Δcolon mice exhibited a 50% reduction in the numbers of serotonin-positive EC cells in the colon, but not the small intestine. Serotonin tissue content in the colon determined by ELISA was also reduced. A microarray analysis of WT versus ZBP-89Δcolon mice revealed an ~4-fold decrease in TPH1 and chromograninA mRNA. Quantitative PCR was used to verify the decrease in ZBP-89 and TPH1 mRNA in 4 mice. Two ZBP-89 consensus elements were identified in the TPH1 promoter. EMSAs and DAPA confirmed ZBP-89 binding to both elements along with Sp1 and Sp3 transcription factors. Mutation of both GC-rich DNA elements in the TPH1-Luc reporter abolished promoter activity. Infecting the ZBP-89Δcolon mice with S. typhi resulted in less submucosal edema than in the ceca of infected WT mice. CONCLUSIONS: ZBP-89 is required for basal TPH1 expression and serotonin production. Reduced TPH1 expression in the ZBP-89Δcolon mice appeared to mitigate the acute mucosal effects of S. typhi infection, presumably due to less serotonin production and fluid secretion.
Su1720 A Novel eADO Biosensor Microelectrode Array (MEA) for Real-Time Electrochemical Detection of eADO Release From Mouse or Human Gut Iveta Grants, Aurosree Bhowmik, Niyati Dhand, Bradley Needleman, Alan Harzman, Jacqueline E. Wunderlich, Fievos L. Christofi Background and Aims: Endogenous adenosine (eADO) release inhibits neurotransmission, modulates gut mucosal reflexes and protects against colitis. ADO is “a danger signal” in diseases and its release is estimated to reach 10-100 fold higher levels than normal. Little is known about release mechanism(s) of eADO and accurate measurement of ADO in extracellular fluid in response to different stimuli is of critical importance in health and disease. The goal was to develop and test a new eADO biosensor to monitor real-time changes of eADO release in gut mouse or human tissues. Methods: An enzyme linked eADO biosensor / microelectrode array (MEA) FAST16 mk electrochemical recording system was used to monitor changes. The MEA tip sensor has S1 and S2 sites to measure local release of eADO and eINO respectively - the eADO platinum(pt) site S1 is coated with 3 enzymes starting with adenosine deaminase (ADA+NP+XO) to sequentially break down ADO to H202 for detection (S2/eINO site lacks ADA). H202 is oxidized on the pt-surface and resulting current is quantified. Standard curves to ADO or INO (0.5-100μM) are used to calculate release (from current of 0.05nA-1.5nA); an ADA inhibitor EHNA (5μM) competitively inhibited ADO responses. Submucosal plexus with mucosa (M- ENSSMP) or ENSSMP is dissected from human sigmoid colon or jejunum (polyp, Roux-en-Y surgeries) or mouse colon. Recordings are made in a perfusion chamber in Krebs buffer at 37°C; 3-5 tissues/ condition are used. A Narishigi (or Eppendorff) micromanipulator visually guided by a stereomicroscope (zoom 1-40X) is used to position the MEA tip on or near the mucosal surface or ganglion (G) to measure rapid release of eADO (or eINO at 40Hz) in a 200μmspace. Results: In mouse or human ENSSMP high K+ depolarization or high Mg2+/low Ca2+ solutions stimulate release of eADO by 0.5-5μM. DMPP (1-10μM) evokes a TTX-insensitive elevation in eADO of 0.25-0.5μM in ENSSMP. Ischemia, 100μM histamine or blockade of nucleoside transport by 2μM NBTI elevates eADO by 0.25-1.0μM. DSS colitis (3%, 7 days) causes low level oscillations in basal eADO release. In human ENSSMP LSM Fluo-4/Ca2+ imaging revealed that NBTI blocks slow synaptic transmission (sEPSCaT) evoked by fiber tract stimulation (n=12 neurons/G). ADO inhibits and ADA (2u/ml) augments - sEPSCaTs (n=20, p<0.01). Touch or mucosal compression (MS) increased eADO release 10-20μM in humucosa >> huENSSMP). DSS colitis augmented eADO release to MS or K+ depolarization in mucosa and caused prolonged, high amplitude/cyclical responses lasting > 20 min (control=5 min). Conclusions: The enzyme-linked eADO biosensor is a suitable technique to monitor realtime release and quantification of eADO in normal or inflamed gut. Release is TTX-insensitive. Further study is needed to distinguish between H202 from eADO release and H202 from inflammation (DK44179-15;DK44179-14S/ARRA).
Su1723 Impaired Gastric Emptying in Gastroesophageal Reflux Disease Rat Models is Caused by a Reduced Response to Ghrelin Hiroshi Takeda, Shuichi Muto, Nobuhiko Oridate, Shunsuke Ohnishi, Koji Nakagawa, Chiharu Sadakane, Yayoi Saegusa, Miwa Nahata, Tomohisa Hattori, Masahiro Asaka Background/Aim: Adequate conclusions on the association between the onset and progression of gastroesophageal reflux disease (GERD) and gastrointestinal motility have not yet been reached; however, some enterokinesis-promoting agents that may reverse the mucosal damage of GERD have been recognized. Furthermore, there is speculation that reduced gastric emptying in GERD patients is partially involved in acid reflux. Ghrelin is a strong appetite-stimulating hormone produced in the stomach, and is known to have a powerful enhancing effect on gastrointestinal motility. We have clarified the involvement of ghrelin in gastric emptying in a GERD rat model. Method: The GERD model was induced in Wistar rats (Omura N et al. Scand J Gastroenterol 1999; 34:948-953). Rat acyl ghrelin was administered intravenously to the GERD model rats, and the effect on gastric emptying using a semi-solid meal method and feeding were examined by comparison with a sham group. Gastric motor activity in the antrum was evaluated using a strain gauge force transducer. Furthermore, in order to study the effects on ghrelin secretory function and signal transmission in the GERD model rats, the ghrelin levels in the blood and expression of appetite-related genes in the hypothalamus were also examined. Results: In comparison with the sham group, gastric emptying was significantly reduced in the GERD model rats (sham rats, 93.4 ± 2.2 %; GERD rats, 59.2 ± 10.9 %; p < 0.05 vs sham rats). In addition, the gastric antral motility index and frequency were reduced in the GERD model rats (Figure).Intravenous (IV) administration of rat acyl ghrelin significantly enhanced gastric emptying in the sham group, but not in the GERD model rats(Table). Furthermore, while IV administration of rat acyl ghrelin enhanced gastric antral motility in the sham group rats, it showed no effect on gastric antral motility in the GERD model rats. Similarly, IV administration of ghrelin to the sham group rats significantly increased feeding (saline, 1.0 ± 0.2 g/h; ghrelin, 1.8 ± 0.2 g/h; p < 0.05), but no effect was observed in the GERD model rats (saline, 0.6 ± 0.1 g/h; ghrelin, 0.4 ± 0.2 g/h). In addition, there was no difference in the plasma GH levels between the two groups of rats. In the GERD model rats, plasma ghrelin levels, hypothalamic NPY/AGRP mRNA expressions were also high compared to the levels in the sham group rats. Conclusion: In the GERD model rats, the ghrelin signal transmission, which may mediate gastric emptying, is reduced. Peripheral ghrelin signals are transmitted to the NPY/AGRP neurons, suggesting the possibility that signals are interrupted after transmission. Table The effect of intravenous administration of rat acyl ghrelin on gastric emptying.
Su1721 Ghrelin Prevents Levodopa-Inhibited Gastric Emptying in Rats Lixin Wang, Andreas Stengel, Miriam Goebel, Marie-Francoise Chesselat, Yvette Tache Background: Levodopa, a precursor of dopamine, is a traditional drug taken orally to treat symptoms of Parkinson's disease (PD). However, it induces delayed gastric emptying (GE), which dampens its absorption in the small intestine associated with motor fluctuation. Levodopa has been administered by different routes in rats and more variations in plasma levels occurred after oral administration compared to intravenous (iv) or intraperitoneal (ip) injection. Ghrelin is a potent prokinetic hormone involved in the regulation of gastric phase III-like contractions and its agonists are in clinical trials for treatment of post-operative ileus. Dopamine and ghrelin have opposite effects on gastrointestinal motility. Aim: To investigate whether ghrelin blocks levodopa inhibitory effect on GE. Methods: GE was determined by the phenol red method in fasted rats 20 min after (1) levodopa (5 or 15 mg/kg) or vehicle (0.3 ml of 0.2N HCl and 7% NaHCO3 at 4:1.5 with 1% ascorbic acid, pH 7) co-administered intragastrically (ig) with a non-nutrient viscous phenol red solution (1.2 ml/rat); (2) ip injection of levodopa (15 mg/kg) or vehicle (0.5 ml/rat) 10 min before ig viscous phenol red solution; (3) iv injection of ghrelin (30 or 100 μg/kg) or vehicle (saline 0.2 ml/rat) 10 min before ig levodopa and viscous phenol red solution; (4) subcutaneous (sc) injection naltrexone (μ opioid receptor antagonist as a control agent, 1 mg/kg) or saline (0.5 ml/rat) 30 min before ig levodopa and viscous phenol red solution. Plasma acyl ghrelin levels after iv levodopa (5 mg/kg) were assessed by radioimmunoassay. Results: Levodopa administered ig dose-dependently decreased GE in fasted rats (5 or 15 mg/kg: 41.0 ± 5.0 or 23.0 ± 4.8% respectively vs. 59.9 ± 3.5% in vehicle, P < 0.05), and the inhibition was more robust after ip injection (15 mg/kg: 8.0 ± 2.5%, P < 0.05). Pretreatment with iv ghrelin completely blocked the ig levodopa-inhibited GE (53.2 ± 3.0 or 59.2 ± 2.3% at 30 or 100 μg/kg vs. 30.1 ± 4.4% with iv saline, P < 0.05) compared to iv saline plus ig vehicle (60.0 ± 5.2%, P > 0.05). Ghrelin increased basal GE (72.9 ± 2.3%) but not significantly compared with saline (P > 0.05). Naltrexone injected sc did not influence the ig levodopa-induced delayed
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AGA Abstracts
AGA Abstracts
the nuclear proteins binding to the TPH1 promoter. ZBP-89Δcolon mice were infected with Salmonella typhimurium (S. typhi) at 107 cfu for 3 days. RESULTS: Although ubiquitously expressed, immunofluorescence confirmed high expression of ZBP-89 protein with serotonin in both mouse and human gut EC cells. ZBP-89Δcolon mice exhibited a 50% reduction in the numbers of serotonin-positive EC cells in the colon, but not the small intestine. Serotonin tissue content in the colon determined by ELISA was also reduced. A microarray analysis of WT versus ZBP-89Δcolon mice revealed an ~4-fold decrease in TPH1 and chromograninA mRNA. Quantitative PCR was used to verify the decrease in ZBP-89 and TPH1 mRNA in 4 mice. Two ZBP-89 consensus elements were identified in the TPH1 promoter. EMSAs and DAPA confirmed ZBP-89 binding to both elements along with Sp1 and Sp3 transcription factors. Mutation of both GC-rich DNA elements in the TPH1-Luc reporter abolished promoter activity. Infecting the ZBP-89Δcolon mice with S. typhi resulted in less submucosal edema than in the ceca of infected WT mice. CONCLUSIONS: ZBP-89 is required for basal TPH1 expression and serotonin production. Reduced TPH1 expression in the ZBP-89Δcolon mice appeared to mitigate the acute mucosal effects of S. typhi infection, presumably due to less serotonin production and fluid secretion.